In order to determine ligand or inhibitor binding constants with proteins in cases not amenable to other means, to study successively more complex protein systems of biological interest and to more deeply understand the theoretical aspects of the method, we are developing quantitative affinity chromatography (QAC) as applied to the general systems of staphylococcal nuclease and bovine pancreatic ribonuclease, their ligands and inhibitors. Elution of highly purified nuclease on an affinity matrix of deoxythymidine-5' -phosphate-3'- (p- aminophenylphosphate)-Sepharose in varying soluble ligand (deoxythymidine-3', 5'-diphosphate) concentration and highly purified ribonuclease on columns of uridine-3'-phosphate-2'-(p- aminophenylphosphate)-Sepharose with varying concentration of cytidine- 2'-phosphate, show competition of soluble ligand with matrix-bound ligand for sites on the protein. In both cases and in those of RNase-S and the ribonuclease semi-synthetic derivative (p-F-phe8 1-15 peptide)S RNase-S', this affinity disruption by soluble ligand is shown to result in enzyme elution at a position universely dependent on ligand concentration and directly so on the appropriate binding constant (Kd, as determined by other methods). The determined elution volume, when used in previously derived equations, permits calculation of constants for protein binding to both soluble and matrix-bound ligand. This technique (QAC) will be applied to other semi-synthetic derivatives of RNase, which, because they lack activity, present a problem in determination of binding constants. Also to be studied are more complex multivalent peptide-protein and protein-protein systems of biological interest.